Mechanisms of eukaryotic transcription activation

描述(申请人提供)：转录激活是许多信号转导和发育途径的最终终点，而了解激活机制是理解这些途径作用的关键。以前的研究表明，通过基因特异性转录激活子的突变扰乱正常的基因调控可能会导致癌症和其他疾病。这项建议的广泛的长期目标是确定基因特异性转录因子通过RNA聚合酶II(POL II)激活转录的机制。这项拟议的工作将为在分子水平上理解正常和疾病状态下的基因调控提供基础。这项工作的具体目标将利用生化、结构和分子遗传学方法来检测几个转录激活剂的直接靶标，以及与这些靶标接触刺激转录的机制。使用一种新开发的方法来绘制大型复合体中的蛋白质-蛋白质接触图，我们将在存在或不存在染色质的情况下识别几个模型激活剂中激活域的直接靶点。将进行生化和分子遗传学研究，以测试这些相互作用在转录中的相关性。我们将扩展这些研究，以检查作用于TATA-less启动子的不同激活剂类别的靶标。在通过羟基自由基裂解和蛋白质-蛋白质直接相互作用分析绘制这些激活剂与靶的相互作用图后，我们将结合相关的靶确定激活域的结构。最后，通过在中间阶段阻断预引发复合体的组装，我们将研究激活剂与其中一个靶标(SAGA共激活复合体)接触刺激转录的机制。我们拟议的工作将阐明转录调控的重要机制和原则。

DESCRIPTION (provided by applicant): Activation of transcription is the ultimate endpoint for many signal transaction and developmental pathways, and understanding the mechanism of activation is a key to understanding the action of these pathways. From previous studies, it is clear that disruption of normal gene regulation by mutations in gene-specific transcription activators can lead to cancer and other diseases. The broad long-term objectives of this proposal are to determine the mechanisms used by gene-specific transcription factors to activate transcription by RNA Polymerase II (Pol II). The proposed work will provide a basis for understanding gene regulation in normal and diseased states at the molecular level. The specific aims of this work will utilize biochemical, structural, and molecular genetic methods to examine the direct targets of several transcription activators and the mechanism whereby contact with these targets stimulates transcription. Using a newly developed method for mapping protein-protein contacts within large complexes, we will identify direct targets of activation domains in several model activators in the presence or absence of chromatin. Biochemical and molecular genetic studies will be conducted to test the relevance of these interactions in transcription. We will extend these studies to examine the targets of a different activator class acting at TATA-less promoters. After mapping these activator-target interactions by hydroxyl radical cleavage and direct protein-protein interaction assays, we will determine the structure of the activation domains in combination with their relevant targets. Finally, by blocking assembly of the Preinitiation Complex at intermediate stages, we will examine the mechanism by which activator contact with 1 of these targets, the SAGA coactivator complex, stimulates transcription. Our proposed work will illuminate important mechanisms and principles of transcriptional regulation.